Reflective mirror-based line-scan adaptive optics OCT for imaging retinal structure and function

Line-scan OCT incorporated with adaptive optics (AO) offers high resolution, speed, and sensitivity for imaging retinal structure and function in vivo. Here, we introduce its implementation with reflective mirror-based afocal telescopes, optimized for imaging light-induced retinal activity (optoretinography) and weak retinal reflections at the cellular scale. A non-planar optical design was followed based on previous recommendations with key differences specific to a line-scan geometry. The three beam paths fundamental to an OCT system –illumination/sample, detection, and reference– were modeled in Zemax optical design software to yield theoretically diffraction-limited performance over a 2.2 deg. field-of-view and 1.5 D vergence range at the eye’s pupil. The performance for imaging retinal structure was exemplified by cellular-scale visualization of retinal ganglion cells, macrophages, foveal cones, and rods in human observers. The performance for functional imaging was exemplified by resolving the light-evoked optical changes in foveal cone photoreceptors where the spatial resolution was sufficient for cone spectral classification at an eccentricity 0.3 deg. from the foveal center. This enabled the first in vivo demonstration of reduced S-cone (short-wavelength cone) density in the human foveola, thus far observed only in ex vivo histological preparations. Together, the feasibility for high resolution imaging of retinal structure and function demonstrated here holds significant potential for basic science and translational applications.     

Design of high-performance adaptive objective lens with large optical depth scanning range for ultrabroad near infrared microscopic imaging

We report on the theory and design of adaptive objective lens for ultra broadband near infrared light imaging with large dynamic optical depth scanning range by using an embedded tunable lens, which can find wide applications in deep tissue biomedical imaging systems, such as confocal microscope, optical coherence tomography (OCT), two-photon microscopy, etc., both in vivo and ex vivo. This design is based on, but not limited to, a home-made prototype of liquid-filled membrane lens with a clear aperture of 8mm and the thickness of 2.55mm ~3.18mm. It is beneficial to have an adaptive objective lens which allows an extended depth scanning range larger than the focal length zoom range, since this will keep the magnification of the whole system, numerical aperture (NA), field of view (FOV), and resolution more consistent. To achieve this goal, a systematic theory is presented, for the first time to our acknowledgment, by inserting the varifocal lens in between a front and a back solid lens group. The designed objective has a compact size (10mm-diameter and 15mm-length), ultrabroad working bandwidth (760nm - 920nm), a large depth scanning range (7.36mm in air) — 1.533 times of focal length zoom range (4.8mm in air), and a FOV around 1mm × 1mm. Diffraction-limited performance can be achieved within this ultrabroad bandwidth through all the scanning depth (the resolution is 2.22 μm - 2.81 μm, calculated at the wavelength of 800nm with the NA of 0.214 - 0.171). The chromatic focal shift value is within the depth of focus (field). The chromatic difference in distortion is nearly zero and the maximum distortion is less than 0.05%.OCIS codes: (220.0220) Optical design and fabrication, (220.1080) Active or adaptive optics, (080.2468) First-order optics, (080.2740) Geometric optical design, (170.3890) Medical optics instrumentation, (170.6900) Three-dimensional microscopy, (170.1790) Confocal microscopy, (170.4500) Optical coherence tomography     

Closed-loop optical stabilization and digital image registration in adaptive optics scanning light ophthalmoscopy

Eye motion is a major impediment to the efficient acquisition of high resolution retinal images with the adaptive optics (AO) scanning light ophthalmoscope (AOSLO). Here we demonstrate a solution to this problem by implementing both optical stabilization and digital image registration in an AOSLO. We replaced the slow scanning mirror with a two-axis tip/tilt mirror for the dual functions of slow scanning and optical stabilization. Closed-loop optical stabilization reduced the amplitude of eye-movement related-image motion by a factor of 10–15. The residual RMS error after optical stabilization alone was on the order of the size of foveal cones: ~1.66–2.56 μm or ~0.34–0.53 arcmin with typical fixational eye motion for normal observers. The full implementation, with real-time digital image registration, corrected the residual eye motion after optical stabilization with an accuracy of ~0.20–0.25 μm or ~0.04–0.05 arcmin RMS, which to our knowledge is more accurate than any method previously reported.OCIS codes: (110.1080) Active or adaptive optics, (120.3890) Medical optics instrumentation, (170.3880) Medical and biological imaging, (170.4470) Ophthalmology, (330.2210) Vision - eye movements     

Scanning,non-contact,hybrid broadband diffuse optical spectroscopy and diffuse correlation spectroscopy system

A scanning system for small animal imaging using non-contact, hybrid broadband diffuse optical spectroscopy (ncDOS) and diffuse correlation spectroscopy (ncDCS) is presented. The ncDOS uses a two-dimensional spectrophotometer retrieving broadband (610-900 nm) spectral information from up to fifty-seven source-detector distances between 2 and 5 mm. The ncDCS data is simultaneously acquired from four source-detector pairs. The sample is scanned in two dimensions while tracking variations in height. The system has been validated with liquid phantoms, demonstrated in vivo on a human fingertip during an arm cuff occlusion and on a group of mice with xenoimplanted renal cell carcinoma.OCIS codes: (170.0170) Medical optics and biotechnology, (170.0110) Imaging systems     

An adaptive optics imaging system designed for clinical use

Here we demonstrate a new imaging system that addresses several major problems limiting the clinical utility of conventional adaptive optics scanning light ophthalmoscopy (AOSLO), including its small field of view (FOV), reliance on patient fixation for targeting imaging, and substantial post-processing time. We previously showed an efficient image based eye tracking method for real-time optical stabilization and image registration in AOSLO. However, in patients with poor fixation, eye motion causes the FOV to drift substantially, causing this approach to fail. We solve that problem here by tracking eye motion at multiple spatial scales simultaneously by optically and electronically integrating a wide FOV SLO (WFSLO) with an AOSLO. This multi-scale approach, implemented with fast tip/tilt mirrors, has a large stabilization range of ± 5.6°. Our method consists of three stages implemented in parallel: 1) coarse optical stabilization driven by a WFSLO image, 2) fine optical stabilization driven by an AOSLO image, and 3) sub-pixel digital registration of the AOSLO image. We evaluated system performance in normal eyes and diseased eyes with poor fixation. Residual image motion with incremental compensation after each stage was: 1) ~2–3 arc minutes, (arcmin) 2) ~0.5–0.8 arcmin and, 3) ~0.05–0.07 arcmin, for normal eyes. Performance in eyes with poor fixation was: 1) ~3–5 arcmin, 2) ~0.7–1.1 arcmin and 3) ~0.07–0.14 arcmin. We demonstrate that this system is capable of reducing image motion by a factor of ~400, on average. This new optical design provides additional benefits for clinical imaging, including a steering subsystem for AOSLO that can be guided by the WFSLO to target specific regions of interest such as retinal pathology and real-time averaging of registered images to eliminate image post-processing.OCIS codes: (110.1080) Active or adaptive optics, (120.3890) Medical optics instrumentation, (170.3880) Medical and biological imaging, (170.4470) Ophthalmology, (330.2210) Vision - eye movements     

Comparison of binocular through-focus visual acuity with monovision and a small aperture inlay

Corneal small aperture inlays provide extended depth of focus as a solution to presbyopia. As this procedure is becoming more popular, it is interesting to compare its performance with traditional approaches, such as monovision. Here, binocular visual acuity was measured as a function of object vergence in three subjects by using a binocular adaptive optics vision analyzer. Visual acuity was measured at two luminance levels (photopic and mesopic) under several optical conditions: 1) natural vision (4 mm pupils, best corrected distance vision), 2) pure-defocus monovision ( + 1.25 D add in the nondominant eye), 3) small aperture monovision (1.6 mm pupil in the nondominant eye), and 4) combined small aperture and defocus monovision (1.6 mm pupil and a + 0.75 D add in the nondominant eye). Visual simulations of a small aperture corneal inlay suggest that the device extends DOF as effectively as traditional monovision in photopic light, in both cases at the cost of binocular summation. However, individual factors, such as aperture centration or sensitivity to mesopic conditions should be considered to assure adequate visual outcomes.OCIS codes: (330.1400) Vision - binocular and stereopsis, (110.1080) Active or adaptive optics, (330.4460) Ophthalmic optics and devices     

Adaptive-optics SLO imaging combined with widefield OCT and SLO enables precise 3D localization of fluorescent cells in the mouse retina

Adaptive optics scanning laser ophthalmoscopy (AO-SLO) has recently been used to achieve exquisite subcellular resolution imaging of the mouse retina. Wavefront sensing-based AO typically restricts the field of view to a few degrees of visual angle. As a consequence the relationship between AO-SLO data and larger scale retinal structures and cellular patterns can be difficult to assess. The retinal vasculature affords a large-scale 3D map on which cells and structures can be located during in vivo imaging. Phase-variance OCT (pv-OCT) can efficiently image the vasculature with near-infrared light in a label-free manner, allowing 3D vascular reconstruction with high precision. We combined widefield pv-OCT and SLO imaging with AO-SLO reflection and fluorescence imaging to localize two types of fluorescent cells within the retinal layers: GFP-expressing microglia, the resident macrophages of the retina, and GFP-expressing cone photoreceptor cells. We describe in detail a reflective afocal AO-SLO retinal imaging system designed for high resolution retinal imaging in mice. The optical performance of this instrument is compared to other state-of-the-art AO-based mouse retinal imaging systems. The spatial and temporal resolution of the new AO instrumentation was characterized with angiography of retinal capillaries, including blood-flow velocity analysis. Depth-resolved AO-SLO fluorescent images of microglia and cone photoreceptors are visualized in parallel with 469 nm and 663 nm reflectance images of the microvasculature and other structures. Additional applications of the new instrumentation are discussed.OCIS codes: (170.4460) Ophthalmic optics and devices, (110.4500) Optical coherence tomography, (110.1080) Active or adaptive optics, (170.0110) Imaging systems, (330.7324) Visual optics, comparative animal models, (170.4470) Ophthalmology     

Longitudinal chromatic aberration of the human eye in the visible and near infrared from wavefront sensing,double-pass and psychophysics

Longitudinal Chromatic Aberration (LCA) influences the optical quality of the eye. However, the reported LCA varies across studies, likely associated to differences in the measurement techniques. We present LCA measured in subjects using wavefront sensing, double-pass retinal images, and psychophysical methods with a custom-developed polychromatic Adaptive Optics system in a wide spectral range (450-950 nm), with control of subjects’ natural aberrations. LCA measured psychophysically was significantly higher than that from reflectometric techniques (1.51 D vs 1.00 D in the 488-700 nm range). Ours results indicate that the presence of natural aberrations is not the cause for the discrepancies across techniques.OCIS codes: (260.0260) Physical optics, (330.0330) Vision, color, and visual optics, (330.4875) Optics of physiological systems, (330.5370) Physiological optics, (220.1010) Aberrations (global), (220.1080) Active or adaptive optics     

Woofer-tweeter adaptive optics scanning laser ophthalmoscopic imaging based on Lagrange-multiplier damped least-squares algorithm

We implemented a Lagrange-multiplier (LM)-based damped least-squares (DLS) control algorithm in a woofer-tweeter dual deformable-mirror (DM) adaptive optics scanning laser ophthalmoscope (AOSLO). The algorithm uses data from a single Shack-Hartmann wavefront sensor to simultaneously correct large-amplitude low-order aberrations by a woofer DM and small-amplitude higher-order aberrations by a tweeter DM. We measured the in vivo performance of high resolution retinal imaging with the dual DM AOSLO. We compared the simultaneous LM-based DLS dual DM controller with both single DM controller, and a successive dual DM controller. We evaluated performance using both wavefront (RMS) and image quality metrics including brightness and power spectrum. The simultaneous LM-based dual DM AO can consistently provide near diffraction-limited in vivo routine imaging of human retina.     

Dual band dual focus optical coherence tomography for imaging the whole eye segment

We developed an improved dual band dual focus spectral domain optical coherence tomography (SD-OCT) for in vivo 2D/3D imaging of the whole eye segment, including the whole anterior segment and retina. The system featured two OCT channels with two different bands centered at 840 nm and 1050 nm, which were designed to image the retina and the anterior segments of the eye, respectively. By combing the two probe light beams for co-axial scanning and separating them for focusing at different segments of the eye with a combination of three dichroic mirrors, we not only minimized the loss of the backscattered light from the sample but also improved the imaging depth, scan range and resolution. The full resolved complex (FRC) method was applied to double the imaging depth for the whole anterior segment imaging, with which an imaging depth of 36.71 mm in air was achieved. We demonstrated that this system was capable of measuring the dynamic changes of ocular dimensions, including the asphericity of the cornea and lens, during accommodation.OCIS codes: (110.4500) Optical coherence tomography, (330.7322) Visual optics, accommodation, (170.3880) Medical and biological imaging, (170.4460) Ophthalmic optics and devices, (170.4470) Ophthalmology, (170.4500) Optical coherence tomography     

Adaptive optics optical coherence tomography at 1 MHz

Image acquisition speed of optical coherence tomography (OCT) remains a fundamental barrier that limits its scientific and clinical utility. Here we demonstrate a novel multi-camera adaptive optics (AO-)OCT system for ophthalmologic use that operates at 1 million A-lines/s at a wavelength of 790 nm with 5.3 μm axial resolution in retinal tissue. Central to the spectral-domain design is a novel detection channel based on four high-speed spectrometers that receive light sequentially from a 1 × 4 optical switch assembly. Absence of moving parts enables ultra-fast (50ns) and precise switching with low insertion loss (−0.18 dB per channel). This manner of control makes use of all available light in the detection channel and avoids camera dead-time, both critical for imaging at high speeds. Additional benefit in signal-to-noise accrues from the larger numerical aperture afforded by the use of AO and yields retinal images of comparable dynamic range to that of clinical OCT. We validated system performance by a series of experiments that included imaging in both model and human eyes. We demonstrated the performance of our MHz AO-OCT system to capture detailed images of individual retinal nerve fiber bundles and cone photoreceptors. This is the fastest ophthalmic OCT system we know of in the 700 to 915 nm spectral band.OCIS codes: (110.1080) Active or adaptive optics, (170.4500) Optical coherence tomography, (120.3890) Medical optics instrumentation, (170.0110) Imaging systems, (170.4470) Ophthalmology, (330.5310) Vision - photoreceptors     

Wavefront sensor-less adaptive optics using deep reinforcement learning

Image degradation due to wavefront aberrations can be corrected with adaptive optics (AO). In a typical AO configuration, the aberrations are measured directly using a Shack-Hartmann wavefront sensor and corrected with a deformable mirror in order to attain diffraction limited performance for the main imaging system. Wavefront sensor-less adaptive optics (SAO) uses the image information directly to determine the aberrations and provide guidance for shaping the deformable mirror, often iteratively. In this report, we present a Deep Reinforcement Learning (DRL) approach for SAO correction using a custom-built fluorescence confocal scanning laser microscope. The experimental results demonstrate the improved performance of the DRL approach relative to a Zernike Mode Hill Climbing algorithm for SAO.     

Digital adaptive optics based on digital lateral shearing of the computed pupil field for point scanning retinal swept source OCT

A novel non-iterative digital adaptive optics technique is presented in which the wavefront error is calculated using the phase difference between the pupil field and its digital copies translated by a pixel along the horizontal and vertical direction in the pupil plane. This method provides slope data per pixel, thus can generate > 50k local slope data samples for a circular pupil of diameter 256 pixels with high accuracy and dynamic range. It offers more than 12x faster computational speed in comparison to the sub-aperture based digital adaptive optics method. Furthermore, it is independent of any system parameters, the light distribution in the pupil plane, or the intensity of the image. The technique is useful in applications such as interferometric or digital holography based microscopy, metrology, and as digital wavefront sensor in adaptive optics, where focusing of light in the sample is involved that creates a guide star or where the sample itself exhibits guide star-like structures. This technique is implemented in a point scanning swept-source OCT at 1060 nm, and a large wavefront error with a peak to valley of 20 radians and root mean square error of 0.71 waves is detected and corrected in case of a micro-beads phantom sample. Also, human photoreceptor images are recovered from aberrated retinal OCT volumes acquired at eccentricities of 2 and 2.5 degrees from the fovea in vivo.     

Simultaneous real-time imaging of the ocular anterior segment including the ciliary muscle during accommodation

We demonstrated a novel approach of imaging the anterior segment including the ciliary muscle using combined and synchronized two spectral domain optical coherence tomography devices (SD-OCT). In one SD-OCT, a Complementary Metal-Oxide-Semiconductor Transistor (CMOS) camera and an alternating reference arm was used to image the anterior segment from the cornea to the lens. Another SD-OCT for imaging the ciliary muscle was equipped with a light source with a center wavelength of 1,310 nm and a bandwidth of 75 nm. Repeated measurements were performed under relaxed and 4.00 D accommodative stimulus states in six eyes from 6 subjects. We also imaged dynamic changes in the anterior segment in one eye during accommodation. The biometry of the anterior segment and the ciliary muscle was obtained. The combined system appeared to be capable to simultaneously real-time image the biometry of the anterior segment, including the ciliary muscle, in vivo during accommodation.OCIS codes: (170.4500) Optical coherence tomography, (170.3880) Medical and biological imaging, (170.4580) Optical diagnostics for medicine, (330.4460) Ophthalmic optics and devices, (330.7322) Visual optics, accommodation     

Anisotropic aberration correction using region of interest based digital adaptive optics in Fourier domain OCT

In this paper a numerical technique is presented to compensate for anisotropic optical aberrations, which are usually present across the lateral field of view in the out of focus regions, in high resolution optical coherence tomography and microscopy (OCT/OCM) setups. The recorded enface image field at different depths in the tomogram is digitally divided into smaller sub-regions or the regions of interest (ROIs), processed individually using subaperture based digital adaptive optics (DAO), and finally stitched together to yield a final image with a uniform diffraction limited resolution across the entire field of view (FOV). Using this method, a sub-micron lateral resolution is achieved over a depth range of 218 μmfor a nano-particle phantom sample imaged using a fiber based point scanning spectral domain (SD) OCM system with a limited depth of focus (DOF) of ~7 μmat a numerical aperture (NA) of 0.6. Thus, an increase in DOF by ~30x is demonstrated in this case. The application of this method is also shown in ex vivo mouse adipose tissue.OCIS codes: (100.2000) Digital image processing, (100.3020) Image reconstruction-restoration, (100.3175) Interferometric imaging, (010.1080) Active or adaptive optics, (110.0180) Microscopy, (110.4500) Optical coherence tomography     

Adaptive optics SLO/OCT for 3D imaging of human photoreceptors in vivo

We present a new instrument that is capable of imaging human photoreceptors in three dimensions. To achieve high lateral resolution, the system incorporates an adaptive optics system. The high axial resolution is achieved through the implementation of optical coherence tomography (OCT). The instrument records simultaneously both, scanning laser ophthalmoscope (SLO) and OCT en-face images, with a pixel to pixel correspondence. The information provided by the SLO is used to correct for transverse eye motion in post-processing. In order to correct for axial eye motion, the instrument is equipped with a high speed axial eye tracker. In vivo images of foveal cones as well as images recorded at an eccentricity from the fovea showing cones and rods are presented.OCIS codes: (170.3890) Medical optics instrumentation, (110.1080) Active or adaptive optics, (170.4470) Ophthalmology, (330.5310) Vision - photoreceptors, (110.4500) Optical coherence tomography     

Integrated adaptive optics optical coherence tomography and adaptive optics scanning laser ophthalmoscope system for simultaneous cellular resolution in vivo retinal imaging

We describe an ultrahigh-resolution (UHR) retinal imaging system that combines adaptive optics Fourier-domain optical coherence tomography (AO-OCT) with an adaptive optics scanning laser ophthalmoscope (AO-SLO) to allow simultaneous data acquisition by the two modalities. The AO-SLO subsystem was integrated into the previously described AO-UHR OCT instrument with minimal changes to the latter. This was done in order to ensure optimal performance and image quality of the AO- UHR OCT. In this design both imaging modalities share most of the optical components including a common AO-subsystem and vertical scanner. One of the benefits of combining Fd-OCT with SLO includes automatic co-registration between two acquisition channels for direct comparison between retinal structures imaged by both modalities (e.g., photoreceptor mosaics or microvasculature maps). Because of differences in the detection scheme of the two systems, this dual imaging modality instrument can provide insight into retinal morphology and potentially function, that could not be accessed easily by a single system. In this paper we describe details of the components and parameters of the combined instrument, including incorporation of a novel membrane magnetic deformable mirror with increased stroke and actuator count used as a single wavefront corrector. We also discuss laser safety calculations for this multimodal system. Finally, retinal images acquired in vivo with this system are presented.     

Contrast-based sensorless adaptive optics for retinal imaging

Conventional adaptive optics ophthalmoscopes use wavefront sensing methods to characterize ocular aberrations for real-time correction. However, there are important situations in which the wavefront sensing step is susceptible to difficulties that affect the accuracy of the correction. To circumvent these, wavefront sensorless adaptive optics (or non-wavefront sensing AO; NS-AO) imaging has recently been developed and has been applied to point-scanning based retinal imaging modalities. In this study we show, for the first time, contrast-based NS-AO ophthalmoscopy for full-frame in vivo imaging of human and animal eyes. We suggest a robust image quality metric that could be used for any imaging modality, and test its performance against other metrics using (physical) model eyes.OCIS codes: (010.1080) Active or adaptive optics, (170.3880) Medical and biological imaging, (170.4460) Ophthalmic optics and devices, (330.4875) Optics of physiological systems     

Fiber-bundle microendoscopy with sub-diffuse reflectance spectroscopy and intensity mapping for multimodal optical biopsy of stratified epithelium

Early detection of structural or functional changes in dysplastic epithelia may be crucial for improving long-term patient care. Recent work has explored myriad non-invasive or minimally invasive “optical biopsy” techniques for diagnosing early dysplasia, such as high-resolution microendoscopy, a method to resolve sub-cellular features of apical epithelia, as well as broadband sub-diffuse reflectance spectroscopy, a method that evaluates bulk health of a small volume of tissue. We present a multimodal fiber-based microendoscopy technique that combines high-resolution microendoscopy, broadband (450-750 nm) sub-diffuse reflectance spectroscopy (sDRS) at two discrete source-detector separations (374 and 730 μm), and sub-diffuse reflectance intensity mapping (sDRIM) using a 635 nm laser. Spatial resolution, magnification, field-of-view, and sampling frequency were determined. Additionally, the ability of the sDRS modality to extract optical properties over a range of depths is reported. Following this, proof-of-concept experiments were performed on tissue-simulating phantoms made with poly(dimethysiloxane) as a substrate material with cultured MDA-MB-468 cells. Then, all modalities were demonstrated on a human melanocytic nevus from a healthy volunteer and on resected colonic tissue from a murine model. Qualitative in vivo image data is correlated with reduced scattering and absorption coefficients.OCIS codes: (120.4640) Optical instruments, (060.2310) Fiber optics, (170.6510) Spectroscopy, tissue diagnostics, (110.2945) Illumination design, (170.2150) Endoscopic imaging     

Improved visualization of outer retinal morphology with aberration cancelling reflective optical design for adaptive optics - optical coherence tomography

We present an aberration cancelling optical design for a reflective adaptive optics - optical coherence tomography (AO-OCT) retinal imaging system. The optical performance of this instrument is compared to our previous multimodal AO-OCT/AO-SLO retinal imaging system. The feasibility of new instrumentation for improved visualization of microscopic retinal structures is discussed. Examples of images acquired with this new AO-OCT instrument are presented.OCIS codes: (110.4500) Optical coherence tomography, (010.1080) Active or adaptive optics, (220.1000) Aberration compensation, (170.0110) Imaging systems, (170.4470) Ophthalmology, (120.3890) Medical optics instrumentation